I have 5 related EST sequences and I want to assemble contigs. I am using the sequence assembly program CAP3. Here is my data:
When I run this through CAP3, I get a contig of EV005953+, EV115686-, and ES981358+, shown below:
However, I saw somewhere else that actually EV115773 should also align with the other sequences. To test this I gave just the relevant 4 sequences as the input:
Now, I get an alignment where all the 4 sequences align:
This seems to be correct. So why did the first run not align EV115773 ut gave it as a singlet? How can we correctly run CAP3 to give consistent results? All of this can be checked using the online CAP3 page at http://pbil.univ-lyon1.fr/cap3.php or by downloading CAP3 from http://seq.cs.iastate.edu/cap3.html
Code:
>ES981358 GTTACTGTCCAGAGATTCGAGTGGGTCCAAAGGGACACAAAGTGAAGATGTGTAAAGCGACAAAACATCAGATGCGAGACGGAATGCACGCGTGGCAGGAAGCGACTATTGACGACGTGGTCGGTCCAAACTATGTTTGGCATGTTCGGGATCCAGATTCTTCTGCTCTTGACAATAGCTTGCAGAGGTTTTATGGGAAAGCTCCTGCGGTTGTGGAGTTGTGTGTGCAAGGTGGTGCACCGGTGCCTGACCAGTACAAGAGTATGATGAGACTCGATGTGGTTTACCCTCAACGTGATGAAGTCGATCTAGTCGCTTGACCTTTCAATGTGATTCAGCATCAAGAAACAATTGATATGAATAAACGAAAGCTCCATATACATATTAGGAAACATAGTTAGATGTTTGATTCTTCTGCTCCGTGCAGCTGTGATCTCACTTCGCAGTTTCGCAGCTTCTCAATAGCTTTGAGGTTAATGCAATTTCAACATTCCCGTTGAGGTTTTGTACCATTAAGGCAAAAATGTTATATAACTTCATAAAAAGTTATAGGAGATGTTTTCTTCCTTTCATATAAACAAAACAAGATTGTGGAATCCATTTTAGTCCAA >EV115773 ACTGNGGTCGCCTGCAGGTACCGGTCTCGGATTCCCGGGTCGACCCACGCGTCCGACCGTTGCCGCGCCTCGTTACTCTCTGGCCTCTCTGCAATCATCCATCACCATGTTCATGTTCATCGCTGCAGATTCTGCTCTGAGGTTCACATTGGTAAGGAAGGTCATGGAATAAGGACTTGCATTGGCCCCGGGAGCGGTTCAAGAAGTGCGACTCATGTATGGAAAAGAGGAAGAGCTAGCGACGTCGTATTGTTCCCAAAATGTTTCCACCTCTATGACCGTGCTGTCAAACCGCGAGTTATCCACGACGAGAGGTTCGCCGTCCCTAAGATCTCTGCGGTTCTTGAGCTATGCATACAGGCAGGCGTGGACATCGATAAGTTCCCAGCGAAGCGAAGATCCAAACCTGTTTACAGCATCGAAGGACGGATCGTAGATTTCGAGGAAGTCAACAACGGGAACTCAGAAACCGCAGTTACTAATTCTACTACTACGGCTACACCTTTACAAGAAGATTATCATTACAGTACAGAGAAGAAGAGCTTGAAGGAGTTAAGCGTTGAGACGATGGACTCATGGTTTGAAATGGTCACAGGGGTCAAGAAACTGATGGAGAGATACAAAGTGTGGACATGTGGTTACTGTCCAGAGATTCAAGTGGGTCCAAAGGGACACAAAGTGAAGATGTGTAAAGCGACAAAACATCAGATGCGAGACGGAATGCACGCGTGGCAAGAAGCGACTATTGACGACGTGGTCGGTCCAAACTATGTTTGGCATGTTCGGGATCCAGATTCTTCTGCTCTTGACATAGCTTGCAGAGGTTTTATGGGAAAGCTCCTGCNGTTGTGGAGTTGTGTGTGCAAGGTGGGTGCACCGTGCCTGACCAGTC >EV115686 GGTATATGAAACCTTTGAACAAACTATTGGACTAAAATGGATTCCACAATCTTGTTTTGTTTATATGAAAGGAAGAAAACATCTCCTATAACTTTTTATGAAGTTATATAACATTTTTGCCTTAATGGTACAAAACCTCAACGGGAATGTTGAAATTGCATTAACCTCAAAGCTATTGAGAAGCTGCGAAACTGCGAAGTGAGATCACAGCTGCACGGAGCAGAAGAATCAAACATCTAACTATGTTTCCTAATATGTATATGGAGCTTTCGTTTATTCATATCAATTGTTTCTTGATGCTGAATCACATTGAAAGGTCAAGCGACTAGATCGACTTCATCACGTTGAGGGTAAACCACATCGAGTCTCATCATACTCTTGTACTGGTCAGGCACCGGTGCACCACCTTGCACACACAACTCCACAACCGCAGGAGCTTTCCCATAAAACCTCTGCAAGCTATTGTCAAGAGCAGAAGAATCTGGATCCCGAACATGCCAAACATAGTTTGGACCGACCACGTCGTCAATAGTCGCTTCTTGCCACGCGTGCATTCCGTCTCGCATCTGATGTTTTGTCGCTTTACACATCTTCACTTTGTGTCCCTTTGGACCCACTTGAATCTCTGGACAGTAACCACATGTCCACACTTTGTATCTCTCCATCAGTTTCTTGACCCCTGTGACCATTTCAAACCATGAGTCCATCGTCTCAACGCTTAACTCCTTCAAGCTCTTCTTCTCTGTACTGTAATGA >EV005953 GAACTCAGAAACCGCAGTTACTAGTGTTAGCACGGCTACACCTTTACAAGAAGATGATCATTACAGTACAGAGAAGAAGAGCTTGAAGGAGTTAAGCGTTAAGACAATGGATTCATGGTTTGAAATGGTCACAGGGGTCAAGGAACTGATGGAGAAATACAAAGTGTGGACTTGTGGTTACTGTCCAGAGGTTCAAGTGGGTCCCAAGGGACACAAAGTGAAGATGTGTAAAGCGACAAAGCATCAGATGCGAGACGGAATGCACGCGTGGCAAGAAGCGACTATTGACGACGTGGTCGGTCCAAACTATGTGTGGCATGTTCGGGATCCAGATTCTTCTGCTCTTGACAATAGCTTGCAGAGGTTTTATGGGAAAGCTCCTGCGGTCGTGGAGTTGTGTGTGCAAGGTGGTGCACCGGTACCTGACCAGTACAAGAGTATGATGAGACTCGATGTGGTTTACCCTCAACGTGATGAAGTCGATCTAGTCGCGTGACCTTTCAAGGTGATTCAGCATCAAGAAACAATTGATATGAAAAAACGAAAGCTCCATATACATATTAGGAAACATAGTTAGATGTTTGGTTCTTCTGCTCCGTGCAGCTTCTCACTTCGCAGCTTCTCAATAGCTTTGAGGTTAATGCAATTTCAACATTCCCGTTGAGGTTTTGTACCA >EE435034 TTATATTGAGGAGATCCCCCGGTGGAGGATGTTAACCGAGATATCTCGGAGAAATATTCGTGCGGGATTTAGCATGATAACGAGGCTCAAAAAATGAAAAGTAGTCTAGCAATTGTAAGCTGAGGACTCTTTCACACAGGAAATAGCTATGATCATGATCATCGCTGGAGATTCTGCTCTGAGGATCACATTGTTTAAGGAAGGTCATGGAATAAGGACTTGCATTGGCCCCGGGAGCGGTTCAAGAAGTGCGACTCATGTATGGAAAAGAGGAAGAGCTAGCGACGTCGTATTGTTCCCAAAATGTTTCCACCTCTATGACCGTGCTGTCAAACCGCGAGTTATCCACGACGAGAGGTTCGCCGTCCCTAAGATCTCTGCGGTTCTTGAGCTATGCATACAGGCAGGCGTGGACATCGATAAGTTCCCAGCGAAGCGAAGATCCAAACCTGTTTACAGCATCGAAGGACGGATCGGTAGATTTCGAGGAAGTCAACAAACGGGAACTCAGAAACCGCAGTTACTAATTCTACTACTACGGCTACACCTTTACAAGAAGATTATCATTACAGTACAGAGAAGAAGAGCTTGAAGGAGTTAAGCGTTGAGACGATGGACTCATGGTTTGAAATGTTCACAGGGGTCA
When I run this through CAP3, I get a contig of EV005953+, EV115686-, and ES981358+, shown below:
Code:
EV005953+ GAACTCAGAAACCGCAGTTACTAGTGTTAGCACGGCTACACCTTTACAAGAAGATGATCA EV115686- TCA ____________________________________________________________ consensus GAACTCAGAAACCGCAGTTACTAGTGTTAGCACGGCTACACCTTTACAAGAAGATGATCA . : . : . : . : . : . : EV005953+ TTACAGTACAGAGAAGAAGAGCTTGAAGGAGTTAAGCGTTAAGACAATGGATTCATGGTT EV115686- TTACAGTACAGAGAAGAAGAGCTTGAAGGAGTTAAGCGTTGAGACGATGGACTCATGGTT ____________________________________________________________ consensus TTACAGTACAGAGAAGAAGAGCTTGAAGGAGTTAAGCGTTAAGACAATGGACTCATGGTT . : . : . : . : . : . : EV005953+ TGAAATGGTCACAGGGGTCAAGGAACTGATGGAGAAATACAAAGTGTGGACTTGTGGTTA EV115686- TGAAATGGTCACAGGGGTCAAGAAACTGATGGAGAGATACAAAGTGTGGACATGTGGTTA ES981358+ GTTA ____________________________________________________________ consensus TGAAATGGTCACAGGGGTCAAGAAACTGATGGAGAAATACAAAGTGTGGACATGTGGTTA . : . : . : . : . : . : EV005953+ CTGTCCAGAGGTTCAAGTGGGTCCCAAGGGACACAAAGTGAAGATGTGTAAAGCGACAAA EV115686- CTGTCCAGAGATTCAAGTGGGTCCAAAGGGACACAAAGTGAAGATGTGTAAAGCGACAAA ES981358+ CTGTCCAGAGATTCGAGTGGGTCCAAAGGGACACAAAGTGAAGATGTGTAAAGCGACAAA ____________________________________________________________ consensus CTGTCCAGAGATTCAAGTGGGTCCAAAGGGACACAAAGTGAAGATGTGTAAAGCGACAAA . : . : . : . : . : . : EV005953+ GCATCAGATGCGAGACGGAATGCACGCGTGGCAAGAAGCGACTATTGACGACGTGGTCGG EV115686- ACATCAGATGCGAGACGGAATGCACGCGTGGCAAGAAGCGACTATTGACGACGTGGTCGG ES981358+ ACATCAGATGCGAGACGGAATGCACGCGTGGCAGGAAGCGACTATTGACGACGTGGTCGG ____________________________________________________________ consensus ACATCAGATGCGAGACGGAATGCACGCGTGGCAAGAAGCGACTATTGACGACGTGGTCGG . : . : . : . : . : . : EV005953+ TCCAAACTATGTGTGGCATGTTCGGGATCCAGATTCTTCTGCTCTTGACAATAGCTTGCA EV115686- TCCAAACTATGTTTGGCATGTTCGGGATCCAGATTCTTCTGCTCTTGACAATAGCTTGCA ES981358+ TCCAAACTATGTTTGGCATGTTCGGGATCCAGATTCTTCTGCTCTTGACAATAGCTTGCA ____________________________________________________________ consensus TCCAAACTATGTTTGGCATGTTCGGGATCCAGATTCTTCTGCTCTTGACAATAGCTTGCA . : . : . : . : . : . : EV005953+ GAGGTTTTATGGGAAAGCTCCTGCGGTCGTGGAGTTGTGTGTGCAAGGTGGTGCACCGGT EV115686- GAGGTTTTATGGGAAAGCTCCTGCGGTTGTGGAGTTGTGTGTGCAAGGTGGTGCACCGGT ES981358+ GAGGTTTTATGGGAAAGCTCCTGCGGTTGTGGAGTTGTGTGTGCAAGGTGGTGCACCGGT ____________________________________________________________ consensus GAGGTTTTATGGGAAAGCTCCTGCGGTTGTGGAGTTGTGTGTGCAAGGTGGTGCACCGGT . : . : . : . : . : . : EV005953+ ACCTGACCAGTACAAGAGTATGATGAGACTCGATGTGGTTTACCCTCAACGTGATGAAGT EV115686- GCCTGACCAGTACAAGAGTATGATGAGACTCGATGTGGTTTACCCTCAACGTGATGAAGT ES981358+ GCCTGACCAGTACAAGAGTATGATGAGACTCGATGTGGTTTACCCTCAACGTGATGAAGT ____________________________________________________________ consensus GCCTGACCAGTACAAGAGTATGATGAGACTCGATGTGGTTTACCCTCAACGTGATGAAGT . : . : . : . : . : . : EV005953+ CGATCTAGTCGCGTGACCTTTCAAGGTGATTCAGCATCAAGAAACAATTGATATGAAAAA EV115686- CGATCTAGTCGCTTGACCTTTCAATGTGATTCAGCATCAAGAAACAATTGATATGAATAA ES981358+ CGATCTAGTCGCTTGACCTTTCAATGTGATTCAGCATCAAGAAACAATTGATATGAATAA ____________________________________________________________ consensus CGATCTAGTCGCTTGACCTTTCAATGTGATTCAGCATCAAGAAACAATTGATATGAATAA . : . : . : . : . : . : EV005953+ ACGAAAGCTCCATATACATATTAGGAAACATAGTTAGATGTTTGGTTCTTCTGCTCCGTG EV115686- ACGAAAGCTCCATATACATATTAGGAAACATAGTTAGATGTTTGATTCTTCTGCTCCGTG ES981358+ ACGAAAGCTCCATATACATATTAGGAAACATAGTTAGATGTTTGATTCTTCTGCTCCGTG ____________________________________________________________ consensus ACGAAAGCTCCATATACATATTAGGAAACATAGTTAGATGTTTGATTCTTCTGCTCCGTG . : . : . : . : . : . : EV005953+ CAGCT EV115686- CAGCTGTGATCTCACTTCGCAGTTTCGCAGCTTCTCAATAGCTTTGAGGTTAATGCAATT ES981358+ CAGCTGTGATCTCACTTCGCAGTTTCGCAGCTTCTCAATAGCTTTGAGGTTAATGCAATT ____________________________________________________________ consensus CAGCTGTGATCTCACTTCGCAGTTTCGCAGCTTCTCAATAGCTTTGAGGTTAATGCAATT . : . : . : . : . : . : EV115686- TCAACATTCCCGTTGAGGTTTTGTACCATTAAGGCAAAAATGTTATATAACTTCATAAAA ES981358+ TCAACATTCCCGTTGAGGTTTTGTACCATTAAGGCAAAAATGTTATATAACTTCATAAAA ____________________________________________________________ consensus TCAACATTCCCGTTGAGGTTTTGTACCATTAAGGCAAAAATGTTATATAACTTCATAAAA . : . : . : . : . : . : EV115686- AGTTATAGGAGATGTTTTCTTCCTTTCATATAAACAAAACAAGATTGTGGAATCCATTTT ES981358+ AGTTATAGGAGATGTTTTCTTCCTTTCATATAAACAAAACAAGATTGTGGAATCCATTTT ____________________________________________________________ consensus AGTTATAGGAGATGTTTTCTTCCTTTCATATAAACAAAACAAGATTGTGGAATCCATTTT . : . : . : . : . : . : EV115686- AGTCCAATAGTTTGTTCAAAGGTTTCATATACC ES981358+ AGTCCAA ____________________________________________________________ consensus AGTCCAATAGTTTGTTCAAAGGTTTCATATACC
Code:
>ES981358 GTTACTGTCCAGAGATTCGAGTGGGTCCAAAGGGACACAAAGTGAAGATGTGTAAAGCGACAAAACATCAGATGCGAGACGGAATGCACGCGTGGCAGGAAGCGACTATTGACGACGTGGTCGGTCCAAACTATGTTTGGCATGTTCGGGATCCAGATTCTTCTGCTCTTGACAATAGCTTGCAGAGGTTTTATGGGAAAGCTCCTGCGGTTGTGGAGTTGTGTGTGCAAGGTGGTGCACCGGTGCCTGACCAGTACAAGAGTATGATGAGACTCGATGTGGTTTACCCTCAACGTGATGAAGTCGATCTAGTCGCTTGACCTTTCAATGTGATTCAGCATCAAGAAACAATTGATATGAATAAACGAAAGCTCCATATACATATTAGGAAACATAGTTAGATGTTTGATTCTTCTGCTCCGTGCAGCTGTGATCTCACTTCGCAGTTTCGCAGCTTCTCAATAGCTTTGAGGTTAATGCAATTTCAACATTCCCGTTGAGGTTTTGTACCATTAAGGCAAAAATGTTATATAACTTCATAAAAAGTTATAGGAGATGTTTTCTTCCTTTCATATAAACAAAACAAGATTGTGGAATCCATTTTAGTCCAA >EV115773 ACTGNGGTCGCCTGCAGGTACCGGTCTCGGATTCCCGGGTCGACCCACGCGTCCGACCGTTGCCGCGCCTCGTTACTCTCTGGCCTCTCTGCAATCATCCATCACCATGTTCATGTTCATCGCTGCAGATTCTGCTCTGAGGTTCACATTGGTAAGGAAGGTCATGGAATAAGGACTTGCATTGGCCCCGGGAGCGGTTCAAGAAGTGCGACTCATGTATGGAAAAGAGGAAGAGCTAGCGACGTCGTATTGTTCCCAAAATGTTTCCACCTCTATGACCGTGCTGTCAAACCGCGAGTTATCCACGACGAGAGGTTCGCCGTCCCTAAGATCTCTGCGGTTCTTGAGCTATGCATACAGGCAGGCGTGGACATCGATAAGTTCCCAGCGAAGCGAAGATCCAAACCTGTTTACAGCATCGAAGGACGGATCGTAGATTTCGAGGAAGTCAACAACGGGAACTCAGAAACCGCAGTTACTAATTCTACTACTACGGCTACACCTTTACAAGAAGATTATCATTACAGTACAGAGAAGAAGAGCTTGAAGGAGTTAAGCGTTGAGACGATGGACTCATGGTTTGAAATGGTCACAGGGGTCAAGAAACTGATGGAGAGATACAAAGTGTGGACATGTGGTTACTGTCCAGAGATTCAAGTGGGTCCAAAGGGACACAAAGTGAAGATGTGTAAAGCGACAAAACATCAGATGCGAGACGGAATGCACGCGTGGCAAGAAGCGACTATTGACGACGTGGTCGGTCCAAACTATGTTTGGCATGTTCGGGATCCAGATTCTTCTGCTCTTGACATAGCTTGCAGAGGTTTTATGGGAAAGCTCCTGCNGTTGTGGAGTTGTGTGTGCAAGGTGGGTGCACCGTGCCTGACCAGTC >EV115686 GGTATATGAAACCTTTGAACAAACTATTGGACTAAAATGGATTCCACAATCTTGTTTTGTTTATATGAAAGGAAGAAAACATCTCCTATAACTTTTTATGAAGTTATATAACATTTTTGCCTTAATGGTACAAAACCTCAACGGGAATGTTGAAATTGCATTAACCTCAAAGCTATTGAGAAGCTGCGAAACTGCGAAGTGAGATCACAGCTGCACGGAGCAGAAGAATCAAACATCTAACTATGTTTCCTAATATGTATATGGAGCTTTCGTTTATTCATATCAATTGTTTCTTGATGCTGAATCACATTGAAAGGTCAAGCGACTAGATCGACTTCATCACGTTGAGGGTAAACCACATCGAGTCTCATCATACTCTTGTACTGGTCAGGCACCGGTGCACCACCTTGCACACACAACTCCACAACCGCAGGAGCTTTCCCATAAAACCTCTGCAAGCTATTGTCAAGAGCAGAAGAATCTGGATCCCGAACATGCCAAACATAGTTTGGACCGACCACGTCGTCAATAGTCGCTTCTTGCCACGCGTGCATTCCGTCTCGCATCTGATGTTTTGTCGCTTTACACATCTTCACTTTGTGTCCCTTTGGACCCACTTGAATCTCTGGACAGTAACCACATGTCCACACTTTGTATCTCTCCATCAGTTTCTTGACCCCTGTGACCATTTCAAACCATGAGTCCATCGTCTCAACGCTTAACTCCTTCAAGCTCTTCTTCTCTGTACTGTAATGA >EV005953 GAACTCAGAAACCGCAGTTACTAGTGTTAGCACGGCTACACCTTTACAAGAAGATGATCATTACAGTACAGAGAAGAAGAGCTTGAAGGAGTTAAGCGTTAAGACAATGGATTCATGGTTTGAAATGGTCACAGGGGTCAAGGAACTGATGGAGAAATACAAAGTGTGGACTTGTGGTTACTGTCCAGAGGTTCAAGTGGGTCCCAAGGGACACAAAGTGAAGATGTGTAAAGCGACAAAGCATCAGATGCGAGACGGAATGCACGCGTGGCAAGAAGCGACTATTGACGACGTGGTCGGTCCAAACTATGTGTGGCATGTTCGGGATCCAGATTCTTCTGCTCTTGACAATAGCTTGCAGAGGTTTTATGGGAAAGCTCCTGCGGTCGTGGAGTTGTGTGTGCAAGGTGGTGCACCGGTACCTGACCAGTACAAGAGTATGATGAGACTCGATGTGGTTTACCCTCAACGTGATGAAGTCGATCTAGTCGCGTGACCTTTCAAGGTGATTCAGCATCAAGAAACAATTGATATGAAAAAACGAAAGCTCCATATACATATTAGGAAACATAGTTAGATGTTTGGTTCTTCTGCTCCGTGCAGCTTCTCACTTCGCAGCTTCTCAATAGCTTTGAGGTTAATGCAATTTCAACATTCCCGTTGAGGTTTTGTACCA
Now, I get an alignment where all the 4 sequences align:
Code:
. : . : . : . : . : . : EV115773+ ACTGNGGTCGCCTGCAGGTACCGGTCTCGGATTCCCGGGTCGACCCACGCGTCCGACCGT ____________________________________________________________ consensus ACTGNGGTCGCCTGCAGGTACCGGTCTCGGATTCCCGGGTCGACCCACGCGTCCGACCGT . : . : . : . : . : . : EV115773+ TGCCGCGCCTCGTTACTCTCTGGCCTCTCTGCAATCATCCATCACCATGTTCATGTTCAT ____________________________________________________________ consensus TGCCGCGCCTCGTTACTCTCTGGCCTCTCTGCAATCATCCATCACCATGTTCATGTTCAT . : . : . : . : . : . : EV115773+ CGCTGCAGATTCTGCTCTGAGGTTCACATTGGTAAGGAAGGTCATGGAATAAGGACTTGC ____________________________________________________________ consensus CGCTGCAGATTCTGCTCTGAGGTTCACATTGGTAAGGAAGGTCATGGAATAAGGACTTGC . : . : . : . : . : . : EV115773+ ATTGGCCCCGGGAGCGGTTCAAGAAGTGCGACTCATGTATGGAAAAGAGGAAGAGCTAGC ____________________________________________________________ consensus ATTGGCCCCGGGAGCGGTTCAAGAAGTGCGACTCATGTATGGAAAAGAGGAAGAGCTAGC . : . : . : . : . : . : EV115773+ GACGTCGTATTGTTCCCAAAATGTTTCCACCTCTATGACCGTGCTGTCAAACCGCGAGTT ____________________________________________________________ consensus GACGTCGTATTGTTCCCAAAATGTTTCCACCTCTATGACCGTGCTGTCAAACCGCGAGTT . : . : . : . : . : . : EV115773+ ATCCACGACGAGAGGTTCGCCGTCCCTAAGATCTCTGCGGTTCTTGAGCTATGCATACAG ____________________________________________________________ consensus ATCCACGACGAGAGGTTCGCCGTCCCTAAGATCTCTGCGGTTCTTGAGCTATGCATACAG . : . : . : . : . : . : EV115773+ GCAGGCGTGGACATCGATAAGTTCCCAGCGAAGCGAAGATCCAAACCTGTTTACAGCATC ____________________________________________________________ consensus GCAGGCGTGGACATCGATAAGTTCCCAGCGAAGCGAAGATCCAAACCTGTTTACAGCATC . : . : . : . : . : . : EV115773+ GAAGGACGGATCGTAGATTTCGAGGAAGTCAACAACGGGAACTCAGAAACCGCAGTTACT ____________________________________________________________ consensus GAAGGACGGATCGTAGATTTCGAGGAAGTCAACAACGGGAACTCAGAAACCGCAGTTACT . : . : . : . : . : . : EV115773+ AATTCTACTACTACGGCTACACCTTTACAAGAAGATTATCATTACAGTACAGAGAAGAAG EV005953+ ACGGCTACACCTTTACAAGAAGATGATCATTACAGTACAGAGAAGAAG EV115686- TCATTACAGTACAGAGAAGAAG ____________________________________________________________ consensus AATTCTACTACTACGGCTACACCTTTACAAGAAGATGATCATTACAGTACAGAGAAGAAG . : . : . : . : . : . : EV115773+ AGCTTGAAGGAGTTAAGCGTTGAGACGATGGACTCATGGTTTGAAATGGTCACAGGGGTC EV005953+ AGCTTGAAGGAGTTAAGCGTTAAGACAATGGATTCATGGTTTGAAATGGTCACAGGGGTC EV115686- AGCTTGAAGGAGTTAAGCGTTGAGACGATGGACTCATGGTTTGAAATGGTCACAGGGGTC ____________________________________________________________ consensus AGCTTGAAGGAGTTAAGCGTTGAGACGATGGACTCATGGTTTGAAATGGTCACAGGGGTC . : . : . : . : . : . : EV115773+ AAGAAACTGATGGAGAGATACAAAGTGTGGACATGTGGTTACTGTCCAGAGATTCAAGTG EV005953+ AAGGAACTGATGGAGAAATACAAAGTGTGGACTTGTGGTTACTGTCCAGAGGTTCAAGTG EV115686- AAGAAACTGATGGAGAGATACAAAGTGTGGACATGTGGTTACTGTCCAGAGATTCAAGTG ES981358+ GTTACTGTCCAGAGATTCGAGTG ____________________________________________________________ consensus AAGAAACTGATGGAGAGATACAAAGTGTGGACATGTGGTTACTGTCCAGAGATTCAAGTG . : . : . : . : . : . : EV115773+ GGTCCAAAGGGACACAAAGTGAAGATGTGTAAAGCGACAAAACATCAGATGCGAGACGGA EV005953+ GGTCCCAAGGGACACAAAGTGAAGATGTGTAAAGCGACAAAGCATCAGATGCGAGACGGA EV115686- GGTCCAAAGGGACACAAAGTGAAGATGTGTAAAGCGACAAAACATCAGATGCGAGACGGA ES981358+ GGTCCAAAGGGACACAAAGTGAAGATGTGTAAAGCGACAAAACATCAGATGCGAGACGGA ____________________________________________________________ consensus GGTCCAAAGGGACACAAAGTGAAGATGTGTAAAGCGACAAAACATCAGATGCGAGACGGA . : . : . : . : . : . : EV115773+ ATGCACGCGTGGCAAGAAGCGACTATTGACGACGTGGTCGGTCCAAACTATGTTTGGCAT EV005953+ ATGCACGCGTGGCAAGAAGCGACTATTGACGACGTGGTCGGTCCAAACTATGTGTGGCAT EV115686- ATGCACGCGTGGCAAGAAGCGACTATTGACGACGTGGTCGGTCCAAACTATGTTTGGCAT ES981358+ ATGCACGCGTGGCAGGAAGCGACTATTGACGACGTGGTCGGTCCAAACTATGTTTGGCAT ____________________________________________________________ consensus ATGCACGCGTGGCAAGAAGCGACTATTGACGACGTGGTCGGTCCAAACTATGTTTGGCAT . : . : . : . : . : . : EV115773+ GTTCGGGATCCAGATTCTTCTGCTCTTGACA-TAGCTTGCAGAGGTTTTATGGGAAAGCT EV005953+ GTTCGGGATCCAGATTCTTCTGCTCTTGACAATAGCTTGCAGAGGTTTTATGGGAAAGCT EV115686- GTTCGGGATCCAGATTCTTCTGCTCTTGACAATAGCTTGCAGAGGTTTTATGGGAAAGCT ES981358+ GTTCGGGATCCAGATTCTTCTGCTCTTGACAATAGCTTGCAGAGGTTTTATGGGAAAGCT ____________________________________________________________ consensus GTTCGGGATCCAGATTCTTCTGCTCTTGACAATAGCTTGCAGAGGTTTTATGGGAAAGCT . : . : . : . : . : . : EV115773+ CCTGCNGTTGTGGAGTTGTGTGTGCAAGGTGG EV005953+ CCTGCGGTCGTGGAGTTGTGTGTGCAAGGTGGTGCACCGGTACCTGACCAGTACAAGAGT EV115686- CCTGCGGTTGTGGAGTTGTGTGTGCAAGGTGGTGCACCGGTGCCTGACCAGTACAAGAGT ES981358+ CCTGCGGTTGTGGAGTTGTGTGTGCAAGGTGGTGCACCGGTGCCTGACCAGTACAAGAGT ____________________________________________________________ consensus CCTGCGGTTGTGGAGTTGTGTGTGCAAGGTGGTGCACCGGTGCCTGACCAGTACAAGAGT . : . : . : . : . : . : EV005953+ ATGATGAGACTCGATGTGGTTTACCCTCAACGTGATGAAGTCGATCTAGTCGCGTGACCT EV115686- ATGATGAGACTCGATGTGGTTTACCCTCAACGTGATGAAGTCGATCTAGTCGCTTGACCT ES981358+ ATGATGAGACTCGATGTGGTTTACCCTCAACGTGATGAAGTCGATCTAGTCGCTTGACCT ____________________________________________________________ consensus ATGATGAGACTCGATGTGGTTTACCCTCAACGTGATGAAGTCGATCTAGTCGCTTGACCT . : . : . : . : . : . : EV005953+ TTCAAGGTGATTCAGCATCAAGAAACAATTGATATGAAAAAACGAAAGCTCCATATACAT EV115686- TTCAATGTGATTCAGCATCAAGAAACAATTGATATGAATAAACGAAAGCTCCATATACAT ES981358+ TTCAATGTGATTCAGCATCAAGAAACAATTGATATGAATAAACGAAAGCTCCATATACAT ____________________________________________________________ consensus TTCAATGTGATTCAGCATCAAGAAACAATTGATATGAATAAACGAAAGCTCCATATACAT . : . : . : . : . : . : EV005953+ ATTAGGAAACATAGTTAGATGTTTGGTTCTTCTGCTCCGTGCAGCT EV115686- ATTAGGAAACATAGTTAGATGTTTGATTCTTCTGCTCCGTGCAGCTGTGATCTCACTTCG ES981358+ ATTAGGAAACATAGTTAGATGTTTGATTCTTCTGCTCCGTGCAGCTGTGATCTCACTTCG ____________________________________________________________ consensus ATTAGGAAACATAGTTAGATGTTTGATTCTTCTGCTCCGTGCAGCTGTGATCTCACTTCG . : . : . : . : . : . : EV115686- CAGTTTCGCAGCTTCTCAATAGCTTTGAGGTTAATGCAATTTCAACATTCCCGTTGAGGT ES981358+ CAGTTTCGCAGCTTCTCAATAGCTTTGAGGTTAATGCAATTTCAACATTCCCGTTGAGGT ____________________________________________________________ consensus CAGTTTCGCAGCTTCTCAATAGCTTTGAGGTTAATGCAATTTCAACATTCCCGTTGAGGT . : . : . : . : . : . : EV115686- TTTGTACCATTAAGGCAAAAATGTTATATAACTTCATAAAAAGTTATAGGAGATGTTTTC ES981358+ TTTGTACCATTAAGGCAAAAATGTTATATAACTTCATAAAAAGTTATAGGAGATGTTTTC ____________________________________________________________ consensus TTTGTACCATTAAGGCAAAAATGTTATATAACTTCATAAAAAGTTATAGGAGATGTTTTC . : . : . : . : . : . : EV115686- TTCCTTTCATATAAACAAAACAAGATTGTGGAATCCATTTTAGTCCAATAGTTTGTTCAA ES981358+ TTCCTTTCATATAAACAAAACAAGATTGTGGAATCCATTTTAGTCCAA ____________________________________________________________ consensus TTCCTTTCATATAAACAAAACAAGATTGTGGAATCCATTTTAGTCCAATAGTTTGTTCAA . : . : . : . : . : . : EV115686- AGGTTTCATATACC ____________________________________________________________ consensus AGGTTTCATATACC
This seems to be correct. So why did the first run not align EV115773 ut gave it as a singlet? How can we correctly run CAP3 to give consistent results? All of this can be checked using the online CAP3 page at http://pbil.univ-lyon1.fr/cap3.php or by downloading CAP3 from http://seq.cs.iastate.edu/cap3.html